Chromosomes must be duplicated for cells to divide. Chromosome replication is performed by a multiprotein machine. The chromosomal duplicating machine of E. coli is the DNA polymerase III holoenzyme. The holoenzyme is composed of 10 different proteins, some in a stoichiometry of two or more, for a total of 17 polypeptide chains. Within this machine are two DNA polymerase III cores for coordinated synthesis of both leading and lagging strands of duplex DNA. The holoenzyme also contains two p "sliding clamps" which encircle DNA and act as mobile tethers to hold the machinery to DNA for highly processive chromosome duplication. The holoenzyme also contains a 5-subunit 7 complex "clamp loader", a circular pentamer that hydrolyzes three ATP to open and close the p ring around primed DNA. This proposal seeks a deep understanding of how DNA polymerase III holoenzyme acts during chromosome duplication. We find that the p clamp binds DNA directly. We plan to identify the residues of p that bind DNA by solving crystal structures of different p-DNA co-crystals, and to study the roles of p residues that bind DNA by mutagenesis. We will also develop chemical inhibitors of p function and solve their cocrystal structure with p. The clamp loader is an asymmetric structure;each of the three ATP sites are in distinct environments. ATP binding and hydrolysis are used to bind p, open the clamp, bind DNA, close p around DNA, and to eject from the p-DNA complex. We plan to determine the individual roles of each ATP site in the clamp loading process. The clamp loader also confers structural asymmetry onto the two polymerases in the holoenzyme and we will determine the positions each core polymerase must occupy to function on either the leading or lagging strands. When the fork encounters a lesion, the high fidelity Pol III will stall. Other DNA polymerases must function with p to bypass the lesion and we will study the process by which two different polymerases bind simultaneously to one p clamp and regulate their control over the single primed terminus. During lagging strand synthesis, the polymerase must rapidly transfer from one p clamp to a new clamp at the next RNA primer. We will study the detailed process by which the lagging strand polymerase recycles on and off p clamps at a replication fork. Chromosome replication is a crucial life process. Hence, the holoenzyme offers an ideal target for antibacterial compounds. Further, cellular replicases from all three domains of life share a similar three component strategy as the E. coli replicase. Hence, the lessons learned from study of E. coli Pol III H.E. should generalize. Finally, replication gone awry form the basis for diseased states, including cancer. Therefore, understanding the basic processes of chromosome duplication may aid drug development.